Ribonucleoprotein (RNP) complexes are integral to cellular function and control of RNP assembly is an important cellular regulatory mechanism and an attractive therapeutic target. One interesting antibiotic target is the RNase P complex that is responsible for 5'-tRNA maturation and, thus, integral to protein synthesis. RNase P is also a tractable system for investigating RNP assembly since in Bacillus subtilis it is composed of a single RNA and a relatively small protein subunit. This proposal addresses experiments designed to gain a more detailed understanding of RNP assembly by investigating the mechanism of RNase P protein folding. Thermodynamic studies using small anionic ligands indicate that P protein folding is a two-state process in which binding and folding are coupled. Binding stoichiometries clearly indicate that two specific sites are responsible for small anion binding/folding. Photoaffinity labeling, mutagenesis and NMR will be used to identify and characterize these specific binding sites and to assess their role in P RNA binding. In addition, amide exchange, 15N relaxation and heteronuclear NMR experiments will be used to characterize changes in conformational dynamics and structure as a function of ligand (small anion vs. RNA). Finally, the kinetic folding pathway for P protein will be determined to gain insight into the requirements for folding in vivo.